miRDB: miRNA binding site prediction software
### Select Custom Prediction from the list of functions on the left.
### 2. For this tutorial, we will select ‘Human’for Species and the ’Sample target mRNA sequence’ as an example, which will auto-populate when selected. ### Importantly, if you input your own sequence from UCSC or anywhere else, use FASTA format. ### This software is forgiving of spaces and numbers input with the FASTA sequence, however,t his program does not allow you to utilise the carat ‘> NM_0000 Sample’ format at the beginning, so leave this out. ### Select ‘Go’ to initiate the prediction.
### 3. When it is finished running, select ‘Retrieve Prediction Results’
### 4. The output file will describe how many predicted miRNAs target the input region. ### THe predicted miRNAs will be ranked in order of Target Score. ### It is important to note that sometimes the outputs of similar sequences will vary a bit in the exact order of miRNAs listed for this reason–verify that the miRNA you compare is in both by examining the entire list–it may be in a different order and still be predicted to be present in the sequence.
### 5. One can click on a desired miRNA for information regarding the specific site locations by clicking the red hyperlink hsa-miR-5011-5p, for example. ### The higher the target score, the more one can trust the prediction. Prediction scores over 80 are thought to be valid. ### This also gives the exact miRNA Sequence, the Seed Location(s) and the input Target Length. ### When analyzing two separate inputs, such as a Wild-type sequence vs. a Mutant sequence with an introduced variation, one can easily compare the number of seed locations found in one vs. the other by comparing these side-by-side.
### 6. Going back to the original Prediction output table, one can instead select the ‘Details’ button to the left of the miRNA of interest. ### This will display useful information regarding both the mature and precursor miRNAs, their sequences, genomic location, and validated and predicted targets. ### It is also helpful to read the References on literature in which the miRNA of interest has been investigated.
### 7. From the ‘Details’ page above, one can click on ‘Predicted Targets’ to see the full list that the miRNA of interest may target. ### Again, these are ranked and given a Target Score to help select those that are most likely to be valid targets.
### 8. For this example, we will select the first Predicted Target by selecting ‘Target Gene’. ### This details all of the Seed Locations within its 3’ UTR, length of the 3’ UTR, the exact miRNA sequence and Target Score. ### It also includes a Gene Description and a helpful ‘NCBI Gene ID’ hyperlink to further investigate the targeted gene.
### 9. Clicking on the ‘NCBI Gene ID’ hyperlink will open a new window with the gene page in the NCBI database. ### From here, one can select ‘FASTA’ directly under the bold heading in order to view the nucleotide sequence.
### 11. In a new browser window, search for ‘UCSC Genome Browser’ and select ‘UCSC Genome Browser Home’
### 12. Then, select ‘Genome Browser’ option (highlighted in green).
### 13. From here, select ‘Human’ and select ‘Dec. 2013 GrCh38(hg38)’ ### Past the accession number obtained from the NCBI FASTA page, and select ‘Go’.
### 14. Select the Dark blue ‘NCBI RefSeq genes’ track (highlighted in green).
### 15. This will display the RefSeq gene information for the target gene. ### Select the ‘mRNA/Genomic Alignment’ (highlighted in green).